Culture of human embryonic cells

ABSTRACT

The invention relates to a method for culturing human embryonic stem cells (hESCs) with a lectin. The invention relates also to the use of a lectin in a method for culturing human embryonic stem cells (hESCs) and a culture medium composition containing a lectin.

FIELD OF THE INVENTION

The invention relates to a method for culturing human embryonic stemcells (hESCs) on a lectin. The invention relates also to the use of alectin in a method for culturing human embryonic stem cells (hESCs) anda culture medium composition containing a lectin attached on theculturing plates.

BACKGROUND OF THE INVENTION

Traditional methods for culturing human embryonic stem cells (hESCs)require the direct use of mouse embryonic fibroblasts (MEFs) as a feederlayer, or feeder-conditioned medium or serum. A medium for a feeder-freeculture of hESCs includes an extracellular matrix extracted from a mousesarcoma and is sold under the trademark Matrigel™ (BD Bioscience, US).Matrigel™ is mostly comprised of laminin and collagen and thesecompounds in purified form have also been tried in culturing hESCs.

Matrigel™ and the other feeder-free media used currently in culturessuffer from xeno contamination, and in addition are subject to largevariability caused by containing growth factors and other undefinedmolecules.

Mallon B. S. et al. have reviewed the attempts made toward xeno-freeculture of hESCs in The International Journal of Biochemistry and CellBiology 38, 1063-1075, 2006. As can be concluded, the culture of hESCssuffers with respect to both technical and clinical potential by the useof cells or extracts originating from animal sources, such as mouseembryonic fibroblasts and an extract from a mouse sarcoma. The currentculture methods are also laborious and difficult to scale. Further, itis often hard to maintain the cells in uniform quality and in anundifferentiated form.

One of the biggest problems of the current methods and media forculturing hESCs arises from the use of animal-derived material in theculture medium.

This problem has now been solved in accordance of the present inventionby providing a method for culturing hESCs using a medium containing alectin as a culturing matrix.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to a method for culturing humanembryonic stem cells (hESC) or a population of hESCs with at least onelectin. The invention is also directed to a culture medium compositioncomprising at least one lectin. Further, the invention is directed tothe use of a lectin in a method for culturing hESCs.

In one embodiment, the invention is directed to method for culturinghESCs with a lectin as a matrix and a definitive, serum- and feeder-freemedium. The invention is also directed to a culture medium compositioncomprising at least one lectin and a definitive, serum- and feeder-freemedium. Further, the invention is directed to the use of a lectintogether with a definitive, serum- and feeder-free media in a method forculturing hESCs.

In one embodiment of the invention the lectin is a natural lectinoriginating and/or derived from a plant or an animal. In anotherembodiment, the lectin is a lectin derivative produced by biotechnologymethods, such as recombinant technology.

In a further embodiment of the invention, the lectin is ECA (sometimesalso called ESL) lectin isolated from Erythrina cristagalli seeds or anessentially similar lectin derivative produced by gene technology means.

The invention is based on the use of at least one lectin, such as aplant lectin, in the culture of hESCs, preferably with a definitive,serum- and feeder-free medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative embodiments of the invention andare not meant to limit the scope of the invention as defined in theclaims in any way.

FIG. 1 shows the colonies of (A); FES 29 cells cultured on ECA-lectinfor 6 passages (original magnification 4×), (B); FES 29 cells duringpassage 14 on ECA (magnification 10×) and (C); FES 30 cells cultured onECA for 7 passages (magnification 10×) obtained in Example 1.

FIG. 2 shows the FACS analysis of the surface markers (A); SSEA3 and (B)Tra-1-60 (or Tra-1-81) expressions on FES 29 cells during ECA culturefrom the beginning of ECA culture (passage 0) to passage 8, and (C)SSEA3 and (1) Tra-1-60 (or Tra-1-81) expressions on FES 30 cells duringECA culture from the beginning of ECA culture (passage 0) to passage 8obtained in Example 1. The surface marker expressions in the controlMatrigel cultures are shown for comparison (p=passage ECA/Matrigel).

FIG. 3 shows the FES 29 cells cultured in suspension for EB formationafter 9 passages on ECA described in Example 1.

FIG. 4 shows the hESC colonies on ECA in StemPro® medium obtained inExample 2: (A) FES 29 cells cultured on ECA for 9 passages: first 7passages in conditioned medium and then 2 passages in StemPro®definitive medium and (8) FES 29 cells during passage 3 in StemPro®medium on ECA passage 10.

FIG. 5 shows the FACS-analysis of the expression of the two surfacemarkers SSEA-3 and Tra-1-60 of undifferentiated hESCs described inExample 2. FES29-cells were cultured on ECA for 10 passages and withStemPro®-medium for the last 3 passages.

FIG. 6 shows the EBs formed from FES 29 cells after 12 ECA passages and4 StemPro® passages obtained in Example 2.

FIG. 7 shows (A) FiPS1-5 and (B-C) FiPS6′-12 cell colonies after 5passages on ECA-lectin in conditioned medium obtained in Example 3.

FIG. 8 shows EBs were formed from FiPS6-12 cells after 6 passages on ECAobtained in Example 3.

FIG. 9 shows a list of lectins whose amino acid sequences are highlyhomologous to that of ECA. Potential N-glycosylation sites have beenindicated with highlighting. Lysine residue, which can be used to linkthe lectin to a surface, have been shown in bolded italics.

DETAILED DESCRIPTION OF THE INVENTION

Human embryonic stem cells (hESCs) are derived from the inner cell massof 3-5 day-old blastocysts. hESCs pose telomerase activity and expresssurface markers SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81. They proliferateon continuous basis when maintained in an appropriate cultureenvironment and differentiate both in vivo and in vitro into endo-,meso- and ectoderm. The differentiation is detected by formation ofembryoid bodies in vitro and teratoma in vivo. hESCs are considered tobe the building blocks for all types of cells in humans and thus havehuge potential in applications of cell therapy and regenerativemedicine. With regard to the safety of the transplantation applicationsof hESCs and the derivatives thereof, it is important to reduce or eveneliminate the xenogenic contamination of these cells.

Induced pluripotent stem (IFS) cells are a type of pluripotent stem cellartificially derived from a non-pluripotent cell, typically an adultsomatic cell by inducing a “forced” expression of certain genes. IPScells are considered to be identical to natural pluripotent stem cells,such as embryonic stem cells in many respects.

In the present invention the term human embryonic stem cell (hESC)refers to natural human embryonic stem cells and IPS cells.

Lectins are sugar-binding proteins. They typically play a role inbiological recognition phenomena involving cells and proteins. Most ofthe lectins are basically non-enzymatic in action and non-immune inorigin. Lectins occur ubiquitously in nature. They may bind to a solublecarbohydrate or to a carbohydrate moiety which is a part of a morecomplex carbohydrate structure, such as a glycoprotein or glycolipid.They typically agglutinate certain animal cells and precipitateglycoconjugates. Lectins serve many different biological functions fromthe regulation of cell adhesion to glycoprotein synthesis and thecontrol of protein levels in the blood. Lectins are also known to playimportant roles in the immune system by recognizing carbohydrates thatare found exclusively on pathogens or that are inaccessible on hostcells. Lectins could be derived from plants, such as legume plants likebeans, grains and seeds. In addition, lectins having an animal originare known. Legume lectins are one of the largest lectin families withmore than 70 lectin family members.

Known lectins isolated from plants are, for example, Con A, LCA, PSA,PCA, GNA, HPA, WGA, PWM, WA, ECA, DSA, UEA-1, PNA, SNA and MAA.Galectins are a family of lectins having mammalian origin. Lectinsrecognizing the “terminal N-acetyllactosamine” structure(Fucα2)_(n)Galβ4GlcNAc, wherein n is 0 or 1, are a group of preferredlectins of the present invention. These lectins include, in particular,ECA (Erythrina cristagalli lectin) and UEA-1 (Ulex europeausagglutinin-I), as well as galectin lectins. In addition, a number ofother natural lectins may have the specificity of recognizing and/orbinding to the “terminal N-acetyllactosamine” structure. Furthermore,natural lectins can be mutagenized to improve their binding or to obtainbinding specificity to the “terminal N-acetyllactosamine”. A list oflectins, whose amino acid sequences are highly homologous to ECA isshown in FIG. 9. These lectins potentially have or may readily bemodified by e.g. mutagenesis to have the same activity as ECA.

In one embodiment of the invention, the lectin is an animal-freegalectin, that is, a recombinant lectin protein produced in cell culturesystem, preferably in a non-animal cell culture system.

In one embodiment of the invention, lectins include alsooligosaccharide-binding protein domains and peptides derived fromlectins. Preferably the lectins do not contain a non-lectin domain, suchas an enzyme domain or toxic domain found, for example, in ricinagglutinin (RCA). The lectins of the present invention further includeany polypeptide or equivalent being functionally a lectin. Antibodiesand oligosaccharide-binding enzymes are examples of the proteins beingfunctional lectins. Preferred enzymes include fucosidases andgalactosidases modified to remove the catalytic activity. The antibodiesinclude all types of natural and genetically engineered variants ofimmunoglobulin proteins. Preferred antibodies include blood group H typeII and terminal N-acetyllactosamine binding antibodies.

In the present invention the term “terminal N-acetyllactosamine” refersto a neutral N-acetyllactosamine with a non-reducing terminal end; theneutral means that the structure is not modified by sialic acid or otheracidic residues. Preferably terminal N-acetyllactosamine isnon-substituted type II N-acetyllactosamine or its α2′-fucosylatedvariant structure (H-type II structure) according to formula(Fucα2)_(n)Galβ4GlcNAc, wherein n is 0 or 1.

The amount of lectin used in a solution is about 0.1-500 μg/ml,preferably about 5-200 μg/ml or about 10-150 μg/ml. The amount of lectinfor immobilization of the cell culture surface is about 0.001-50 μg/cm²,preferably from about 0.01-50 μg/cm² to about 0.1-30 μg/cm², morepreferably about 0.3-10 μg/cm² for a lectin with Mw of about 50 kDa, orcorresponding molar density per surface area used. In one embodiment,about 1-50 μg/cm², or about 5-40 μg/cm², preferably about 1040 μg/cm² oflectin is used in a solution to coat a plastic cell culture surface. Inone embodiment, the concentration in the coating solution is betweenabout 50-200 μg/ml for a lectin with Mw of about 50 kDa or correspondingmolar density per surface area used. In a specific embodiment, a plasticcell culture well with polystyre surface is coated by passive adsorbtionusing about 140 μg/ml solution in amount of about 30 μg/cm² for a lectinwith Mw of about 50 kDa.

The present invention relates to a method for culturing human embryonicstem cells (hESC) or a hESC polulation with a lectin. The invention isalso directed to a culture medium composition comprising a lectin as amatrix. Further, the invention is directed to the use of a lectin in amethod for culturing hESCs.

In one embodiment, the invention is directed to a method for culturinghESCs with at least one lectin and to a culture medium compositioncomprising at least one lectin. Further, the invention is directed tothe use of at least one lectin in a method for culturing hESCs.

In one embodiment of the invention the lectin is a natural plant lectinsuch as ECA lectin and in another embodiment of the invention at feastone of the lectins is ECA lectin.

In one embodiment, the invention is directed to method for culturinghESCs with a lectin as a culturing matrix and a definitive, serum- andfeeder-free medium. The invention is also directed to a culture mediumcomposition comprising a lectin and a definitive, serum- and feeder-freemedium. Further, the invention is directed to the use of a lectintogether with a definitive, serum- and feeder-free media in a method forculturing hESCs.

In another embodiment, the invention is directed to method for culturinghESCs with at least one lectin and a definitive, serum- and feeder-freemedium and to a culture medium composition comprising at least onelectin and a definitive, serum- and feeder-free medium. Further, theinvention is directed to the use of at least one lectin together with adefinitive, serum- and feeder-free media in a method for culturinghESCs.

A definitive or fully-defined, serum- and feeder-free medium is a mediumthat is specifically formulated for the uniform growth of hESCs andcontains ingredients required for maintaining normal morphology,pluripotency and differentiation capability of hESCs. StemPro® hESC SFM,developed and sold by Invitrogen Corporation, US, is an example of thiskind of a definitive, serum- and feeder-free medium developed forculturing of hESCs without feeder cells.

In a further embodiment of the invention, the definitive, serum- andfeeder-free medium is StemPro®hESC SFM.

According to the present invention, a lectin is used as a sole culturematrix ingredient or it is added to a culture media applicable to thegrowth of hESCs or used with such a medium. The culture media can alsobe supplemented, for example, with a single or a plurality of growthfactors selected from, for example, a WNT signaling agonist, TGF-b,bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11, IL-5,Flt-3/Flk-2 ligand, fibronectin, LIF, I-IGF, NFG, angiopoietin-like 2and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre, or a mixture thereof.

In one embodiment of the invention, the hESCs are grown on a lectin,such as a plant lectin or galectin coated plate or vessel.

The hESCs cultured according to the present invention are not exposed toanimal-derived material during their cultivation, at least not in suchan extent than cells cultured according to the known methods usingfeeder cells, Matrigel™ and/or other animal-derived material.

The hESCs cultured according to the present invention have shown to havethe typical characteristics of human embryonal stern cells, posingtelomerase activity and expressing surface markers SSEA-3, Tra-1-60 andTra-1-81. In addition, the cells have been shown to be able todifferentiate by forming embryoid bodies and/or teratomas.

The method and the culture medium composition of the present inventionprovide means for culturing hESCs substantially free of xenogeniccontamination. The human embryonic stem cell(s) and/or cellpopulation(s) cultured according to the present invention are thus safefor the current and future transplantation applications.

The following examples represent illustrative embodiments of theinvention without limiting the invention any way.

Example 1 Human Embryonic Stem Cell (hESC) Lines Cultured on ECA-LectinCoated Plastic Generation and Maintenance of hESC Lines

Processes for generation of hESC lines from blastocyst stage of in vitrofertilized human embryos have been described previously in Thomson etal. (Science, 282:1145-1147, 1998). Cell lines FES 22, FES 29 and FES 30were initially derived and cultured either on mouse embryonicfibroblasts feeders (MEFs; 12-13 pc fetuses of the ICR strain), or onhuman foreskin fibroblast feeder cells (HFFs; CRL-2429 ATCC, Mananas,USA) (Mikkola et al. BMG Dew Biol, 6:40-51, 2006). All the lines werecultured in serum-free medium (KnockOut™ D-MEM; Gibco® Cell culturesystems, Invitrogen, Paisley, UK) supplemented with 2 mML-Glutamin/Penicillin streptomycin (Sigma-Aldrich), 20% KnockOut SerumReplacement (Gibco), 1× non-essential amino acids (Gibco), 0.1 mM3-mercaptoethanol (Gibco), 1×ITS (Sigma-Aldrich) and 4 ng/ml bFGE(Sigma/Invitrogen) on feeder cells, or on Matrigel™ (BD Biosciences) inthe same medium (supplemented with additional 4 ng/ml bFGF) conditionedover night on MEFs. Passaging was done either mechanically orenzymatically using collagenase IV (Gibco).

ECA-Lectin Coating of Cell Culture Plates

ECA-lectin (EY laboratories, USA) was dissolved in phosphate bufferedsaline 140 μg/ml. Lectin dilution was sterile filtrated using Millex-GVsyringe driven filter units (0.22 μm, SLGV 013 SL, Millipore, Ireland)and allowed to passively adsorb on cell culture plate by overnightincubation at +4° C. After incubation the wells were washed three timeswith phosphate buffered saline and stem cells were plated on them.

hESC Culturing on ECA-Lectin Coated Cell Culture Plates

The hESC lines (FES 22, FES 29, FES 30) were cultured at least threepassages on Matrigel™ before transferring them onto ECA coated plates,FES 29 was transferred also directly from MEFs onto ECA coated plates inconditioned medium. All lines were maintained on Matrigel™ as controls.The growing cell aggregates were then passaged to new plates at 3-7 dayintervals.

hESC Embryoid Body (EB) Formation

EBs were generated as previously described in Mikkola et al. (BMC DevBiol, 6:40-51, 2006) with small modifications. Briefly, to induce theformation of EBs the confluent hESC colonies were first treated with 200U/ml collagenase IV and transferred on non-adherent Petri dishes to formsuspension cultures. The formed EBs were cultured in suspension for thenext 10 days in standard culture medium (see above) without bFGF.

Teratoma Assay

In order to study teratoma formation about 200 000 morphologically goodlooking hESCs were injected into the testes of nude mice. The resultingtumors wore harvested 8 weeks later and fixed with formalin forimmunohistological examination (Mikkola et al. BMC Dev Biol, 6:40-51,2006).

Flow Cytometry

hESCs were detached enzymatically and washed in 1% ultra pure BSA inPBS. Monoclonal antibodies against SSEA-3, Tra-1-60 and Tra-1-81 (1:50;gifts kindly provided by ESTOOLS www.estools.org) were used as markersfor undifferentiated hESCs. Staining was performed according tomanufacturer's instructions, FACS analysis was done with FACS Caliburmachinery and CellquestPro software (Becton Dickinson).

Results

Three different hESC-lines, FES 22, FES 29 and FES 30, were cultured onECA-coated wells in MEF-conditioned medium up to 23 passages. Themorphology of hESCs was similar to the control Matrigel cultures andhESCs looked undifferentiated after ECA-lectin passages (FIG. 1). LinesFES 29 and FES 30 were repeatedly successfully transferred from Matrigelto ECA-plates. FES 29 cells were also transferred onto ECA-lectinstraight from feeder cells (MEFs).

The expression of surface markers of undifferentiated hESCs (SSEA-3 andTra-1-60/Tra-1-81) were analyzed every 2 or 3 passages by flowcytometry. The follow-up of the surface marker expression during theculture of FES 29 and FES 30 cells on ECA is shown in FIG. 2.

The pluripotency of hESCs after several ECA passages was verified bytheir ability to form EBs in suspension culture or teratomas in nudemice. FES 30 cells cultured 23 passages on ECA and FES 29 Cells cultured4 passages on ECA formed teratoma-containing tissues from all three germcell layers (data not shown). EBs were successfully formed from FES 29and FES 30 cells after ECA-culture (FIG. 3).

Example 2 Culturing hESCs on ECA Lectin in Definitive Medium CulturinghESCs

The FES 29 hESC line (see example 1) was cultured 14 passages onMatrigel™ before transferring the cells on ECA-lectin coated plates. ThehESCs were cultured on ECA-lectin coated plates for 7 passages inMEF-conditioned medium and then changed to a definitive medium, StemPro®hESC SFM (Gibco, Invitrogen A10007-01). Matrigel™ was used as a control.For enzymatic passaging the cells were exposed to 200 units/mlcollagenase IV (Gibco) for 1-2 min at 37° C., washed once in PBS enddissociated by gently pipetting and plated on 2-3 new dishes.

hESC Embryoid Body (EB) Formation

EBs were generated as previously described in Mikkola et al. (2006).Briefly, to induce the formation of EBs the confluent hESC colonies werefirst treated with 200 U/ml collagenase IV and then transferred onnon-adherent Petri dishes to form suspension cultures. The formed EBswere cultured in suspension for the next 10 days in the standard culturemedium (see above) without bFGF.

Flow Cytometry

hESCs were detached enzymatically and washed with 1% ultra pure BSA inPBS. Monoclonal antibodies against SSEA-3, Tra-1-60 and Tra-1-81 (1:50;gifts kindly provided by ESTOOLS www.estools.org) were used as markersfor undifferentiated hESCs. Staining was performed according tomanufacturer's instructions. FACS analysis was done with FACS Caliburmachinery and CellQuestPro software (Becton Dickinson).

Results

hESCs, FES 29 line, were cultured on ECA-lectin for 7 passages withMEF-conditioned culture medium. In the 8^(th) passage conditioned mediumwas changed to the commercial definitive medium, StemPro® hESC SFM. FES29 cells maintained their undifferentiated state and pluripotency duringup to 5 passages in definitive medium on ECA. In FIG. 4 the typical hESCcolonies on ECA in the StemPro® medium are shown. FACS-analysis of theexpression of surface markers of undifferentiated hESCs (SSEA-3 andTra-1-60) is presented in FIG. 5. EBs were formed after 12 passages onECA and 4 passages in the StemPro® medium (FIG. 8).

Example 3 Culturing Induced Pluripotent Stem (IPS) Cells on ECA LectinCoated Plastic Culturing IPS Cells

Two lines of IPS cells were originated either from human embryonal lungfibroblasts or human foreskin fibroblasts with protocol modified fromOkita et al. (Nature, 448:313-317, 2007) and Wernig et al. (Nature,448:318-324, 2007). FiPS1-5 and FiPS6-12 lines were cultured for 10 or 8passages on MEFs before transferring them onto ECA or Matrigel™ coateddishes in MEF-conditioned medium. For enzymatic passaging the cells weresubjected to 200 units/ml collagenase IV for 1-2 min at 37° C., washedonce in PBS and dissociated by gently pipetting and plated on 2-3 newdishes.

hESC Embryoid Body (EB) Formation

EBs were generated as previously described in Mikkola et al. (2006).Briefly, to induce the formation of EBs the confluent cell colonies werefirst treated with 200 U/ml collagenase IV and then transferred onnon-adherent Petri dishes to form suspension cultures. The formed EBswere cultured in suspension for the next 10 days in standard culturemedium (see above) without bFGF.

Results

IPS-cells were cultured in similar in vitro conditions as hESCs. Two IPScell-lines, FiPS1-5 and FiPS6-12, were transferred from MEFs (afterpassage 10 or 8, respectively) to Matrigel or to ECA-coated plates inMEF-conditioned medium. IPS cells were morphologically similar to hESCsin all culturing conditions (FIG. 7). EBs were formed from FiPS6-12cells after 6 passages on ECA (FIG. 8).

1. A method for culturing human embryonal stem cells (hESCs) wherein acell or a cell population is contacted with at least one lectin.
 2. Themethod of claim 1 wherein a cell or a cell population is contacted withat least one lectin and with a definitive serum- and feeder-free medium.3. The method of claim 1 or 2 wherein a cell or a cell population iscontacted with one lectin.
 4. The method of claim 1 wherein the lectinrecognizes the structure (Fucα2)_(n)Galβ4GlcNAc, wherein n is 0 or
 1. 5.The method of claim 4 wherein the lectin is ECA, galectin, or anessentially similar protein biotechnologically produced thereof.
 6. Themethod of claim 2 wherein the definitive, serum- and feeder-free mediumis StemPro® hESC SFM.
 7. A culture medium composition for culturinghuman embryonal stem cells (hESCs), wherein the composition comprises atleast one lectin as a matrix.
 8. The composition of claim 7 wherein itcomprises in addition to at least one lectin as a matrix, a definitiveserum and feeder-free medium.
 9. The composition of claim 7 or 8 whereinit comprises one lectin.
 10. The composition of claim 7, wherein thelectin recognizes the structure (Fucα2)_(n)Galβ4GlcNAc, wherein n is 0or
 1. 11. The composition of claim 10 wherein the lectin is ECA,galectin, or an essentially similar protein biotechnologically producedthereof.
 12. The composition of claim 8 wherein the definitive, serum-and feeder-free medium is StemPro® hESC SFM.
 13. Use of one or morelectins in a method for culturing hESCs.
 14. The use of claim 13 whereinone or more lectins is used as a matrix together with a definitiveserum- and feeder-free medium.
 15. The use of claim 13 or 14 wherein oneor more lectins recognizes the structure (Fucα2)_(n)Galβ4GlcNAc, whereinn is 0 or
 1. 16. The use of claim 15 wherein at least one of the lectinsis ECA, galectin, or an essentially similar protein biotechnologicallyproduced thereof.
 17. The use of claim 14 wherein the definitive, serum-and feeder-free medium is StemPro® hESC SFM.